EP3458844B1 - Procede et dispositif permettant d'enregistrer des parametres de processus de cultures liquides - Google Patents

Procede et dispositif permettant d'enregistrer des parametres de processus de cultures liquides Download PDF

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Publication number
EP3458844B1
EP3458844B1 EP17757504.0A EP17757504A EP3458844B1 EP 3458844 B1 EP3458844 B1 EP 3458844B1 EP 17757504 A EP17757504 A EP 17757504A EP 3458844 B1 EP3458844 B1 EP 3458844B1
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EP
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Prior art keywords
microreactors
excitation light
optical
liquid culture
emission spectrum
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German (de)
English (en)
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EP3458844A1 (fr
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Jochen Büchs
Tobias Ladner
Georg Wandrey
Oliver Paquet-Durand
Bernd Hitzmann
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Rheinisch Westlische Technische Hochschuke RWTH
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Rheinisch Westlische Technische Hochschuke RWTH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/16—Vibrating; Shaking; Tilting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
    • B01F31/20—Mixing the contents of independent containers, e.g. test tubes
    • B01F31/22—Mixing the contents of independent containers, e.g. test tubes with supporting means moving in a horizontal plane, e.g. describing an orbital path for moving the containers about an axis which intersects the receptacle axis at an angle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/08—Optical fibres; light guides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/12—Circuits of general importance; Signal processing
    • G01N2201/129—Using chemometrical methods
    • G01N2201/1293—Using chemometrical methods resolving multicomponent spectra

Definitions

  • the invention relates to a method and a device for recording process parameters by means of 2D fluorescence spectroscopy in liquid cultures in several microreactors of at least one microtiter plate, which is continuously shaken with an orbital shaker at least until the end of the reaction in all microreactors.
  • Methods and devices for recording process parameters in microbial cultures in several microreactors of a microtiter plate, which is continuously shaken with an orbital shaker at least until the reaction in all microreactors is complete are from the EP 1 730 494 B1 known.
  • the known method is particularly suitable for the automated recording of process parameters of microbial cultures, which are shaken without interruption in all microreactors until the reaction is complete. For example, the fluorescence of fluorescent proteins or amino acids and the light backscatter as a measure of the biomass concentration are recorded as process parameters.
  • the ones from the EP 1 730 494 B1 known device for carrying out the method has a microreactor platform connected to an orbital shaker, on which a microtiter plate with a transparent bottom is arranged, which is continuously orbitally shaken until the reaction in all microreactors is complete.
  • the process parameters are recorded using a Sensor optics that include a light-sensitive sensor, a light source and at least one optical waveguide.
  • One end of the optical waveguide is moved from microreactor to microreactor below the microtiter plate using an xy positioning unit.
  • the light from a light source is coupled in, while a light sensor is attached to the other strand of the y-shaped optical waveguide.
  • the optical waveguide is not moved during the acquisition of the process parameters, so that the shaken microreactors move relative to the optical waveguide.
  • the resulting relative movement between the microreactors and the fixed optical waveguide for the measurement is unproblematic as long as the electromagnetic radiation is introduced exclusively into a single one of the microreactors and the electromagnetic radiation (emission) emanating from the microreactor as a result of the introduced electromagnetic radiation (excitation light) exclusively from the Light sensor of the sensor optics is detected.
  • the introduced electromagnetic radiation is introduced with a certain excitation wavelength. Consequently, with the known method only process parameters of substances can be recorded which show fluorescence activity on the radiated electromagnetic radiation or backscatter.
  • a Horiba Fluoromax 4P was used for the measurement in order to scan a very narrow wave range of 500-550 nm around the excitation maximum.
  • the Horiba Fluoromax 4P has a light source and a rotatable monochromator with which light of different wavelengths can be passed through a cuvette. On the emission side, the Horiba Fluoromax 4P also has a rotatable monochromator, which directs the desired wavelength to a light sensor (photomultiplier) (cf. " FluoroMax-4 & FluoroMax-4P with USB (excerpt) ", Operation Manual, Part number J810005 rev.
  • KENSY FRANK ET AL "Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations", MICROBIAL CELL FACTORIES, Vol. 8, No. 1, December 22 2009 (2009-12-22), page 68, XP021067676, ISSN: 1475-2859 also describe fermentation in a BioLector with a fixed excitation wavelength of 485 nm and a shaking frequency from 995 rpm. Furthermore, the basic possibility of installing a fluorescence sensor on a BioLector is disclosed, which is described in the EP 1 730 494 B1 is described.
  • the invention is based on the object of creating a method which allows process parameters to be recorded even for substances which themselves have no fluorescence activity. Furthermore, the method should enable the determination of the physiological state of the liquid culture and of substance transfer rates with relatively little effort and in a short time. Finally, a device for carrying out the method is to be specified.
  • the solution to this problem is based on the idea of recording 2D fluorescence spectra in a large number of, in particular, different liquid cultures in microreactors of shaken microtiter plates.
  • the object is achieved by a method with the features of claim 1 and by a device with the features of claim 27.
  • a measurement i.e. a 2D fluorescence spectrum, could only be recorded every 20.2 hours for each microreactor. This time interval is clearly too long to monitor parallel cultivations in microtiter plates online.
  • the recording of a complete 2D fluorescence spectrum in the excitation range of 250-730 nm with an excitation step size of 10 nm can be reduced to 24 seconds per microreactor.
  • a complete microtiter plate comprising 48 microreactors can therefore be measured in 30 minutes.
  • the monochrome excitation light is generated in an advantageous embodiment of the invention with an automatically tunable monochromator for the spectral isolation of different wavelengths from the incident light of a light source.
  • the transmission and introduction of the excitation light into the individual microreactors as well as the transmission of the emission spectrum from the microreactor to the optical element takes place with the aid of a beam guidance system.
  • the beam guidance system comprises an optical coupling, at least one optical waveguide and, if necessary, further optical elements, such as, for example, semi-transparent mirrors.
  • the optical coupling set up to introduce the excitation light into the liquid culture of a microreactor and to couple the emission spectrum from the liquid culture into the beam guidance system, can be formed, for example, by an end of one or more optical waveguides aligned with the microreactor.
  • lenses or lens arrangements can form the optical coupling.
  • the coupling is not moved during the recording of the 2D fluorescence spectrum, so that the microreactor moves relative to the optical coupling. After the 2D fluorescence spectrum has been recorded, the optical coupling is moved to another microreactor.
  • the microreactors preferably do not have any strong flow disturbances or a round cross section.
  • microtiter plates can be placed on a microreactor platform are attached, the microreactors of all microtiter plates being measured sequentially with a measuring device.
  • the coupling of the measuring device is not only moved from microreactor to microreactor below a single microtiter plate with the xy positioning unit, but also below several microtiter plates. For example, if four microtiter plates with 48 microreactors are attached to a microreactor platform, a total of 192 parallel cultivations can be carried out.
  • the time interval between two measurements of the same microreactor increases with the number of parallel cultivations.
  • several measuring devices are therefore provided in one embodiment of the invention, which allow the simultaneous recording of 2D fluorescence spectra in different microreactors. If the optical coupling of the multiple measuring devices with a positioning unit is moved between the microreactors of one or more microtiter plates, the different microreactors can be approached synchronously and each coupling underneath the microtiter plate can be aligned accordingly. In this embodiment of the invention, 2D fluorescence spectra can be recorded simultaneously in several microreactors of one or more microtiter plates.
  • Another possibility to shorten the time for the measurement of the liquid cultures of a complete microtiter plate is to measure several microreactors without repositioning the coupling.
  • the shaking diameter of the orbital shaker becomes this Purpose coordinated in such a way that several microreactors circle one after the other over the optical coupling during one rotation of the orbital shaker, the recorded fluorescence spectra being assigned to the microreactors circulating over the optical coupling.
  • the time of the measurement and the shaker position are synchronized.
  • the position of the liquid culture in the microreactor depends on the position of the shaker.
  • the introduction of the excitation light is interrupted depending on the position of the orbital shaker.
  • the interruption takes place in particular by means of a shutter.
  • the shutter is an opaque, mechanically movable element for interrupting the optical path of the excitation light.
  • the light source for generating the excitation light can also be clocked as a function of the shaker position.
  • the position of the orbital shaker is determined with a position encoder. For example, a Hall sensor and a magnet attached to the shaft of the orbital shaker can be used to determine the position.
  • a light barrier can be used.
  • An essential advantage of the introduction of the excitation light, which is synchronized with the position of the orbital shaker, is that clearly defined areas of the microreactor can be measured, for example the sickle of liquid of the liquid culture that forms during the shaking movement.
  • each Measurement of reflected excitation light recorded by the sensor matrix is therefore masked out in the emission spectrum.
  • the continuously changing excitation wavelength can preferably be guided past the sensor matrix by specifically changing the position of the optical element that area of the emission spectrum whose wavelength is less than or equal to the excitation wavelength.
  • the excitation wavelength can be set with a movable diaphragm between the optical element and the sensor matrix be hidden.
  • the masking of the excitation wavelength provided according to the invention enables higher intensities of the excitation light.
  • the greater excitation intensity leads to emission spectra with stronger fluorescence signals and ultimately to improved measured values (2D fluorescence spectra).
  • the excitation light is parallelized or focused before it is introduced into the liquid culture.
  • a collimator in particular, can be considered as the lens for parallelizing the excitation light.
  • the higher energy level of the excitation light is particularly advantageous if, during one revolution of the orbital shaker, a measuring device revolves in several circles above the coupling Microreactors a 2D fluorescence spectrum is to be recorded.
  • the emission spectrum to be coupled into the beam guidance system, in particular the optical waveguide can be bundled.
  • the backscattering of the excitation light radiated into the liquid culture is detected with a separate light-sensitive sensor of the measuring device.
  • Information about the growth behavior or the morphology of microorganisms during the reaction in the culture medium of the liquid culture can be obtained from the backscatter.
  • the light scattering is preferably transmitted to the light-sensitive sensor via a further optical waveguide.
  • the optical waveguide for the introduction of the excitation light and the further optical waveguide for the transmission of the backscatter are preferably arranged with respect to one another in the coupling of the beam guidance system that the focus of both optical waveguides lies in one point.
  • This arrangement has the advantage that no realignment of the coupling is required to measure the backscatter, and both measurements can therefore be carried out at shorter time intervals.
  • the optical waveguide can be arranged in such a way that the scattered light guided past the sensor matrix by means of the movable optical element or scattered light reflected by the diaphragm is coupled into one end of the optical waveguide.
  • the beam guidance system can have two separate optical waveguides, one for the excitation light and one for the emission spectrum or a y-optical waveguide with separate fibers for the excitation light and the emission spectrum.
  • the excitation light is deflected by a semi-transparent mirror and introduced into the liquid culture via an optical waveguide with only one fiber.
  • the emission spectrum is transmitted in the beam guidance system through the one optical waveguide and the semitransparent mirror to the optical element that fans out the emission spectrum onto the sensor matrix.
  • the radius of the one optical waveguide can be reduced overall compared to the two-fiber y-optical waveguide and thus the excitation radiation can be better focused.
  • Cross-talk with neighboring microreactors can be prevented.
  • samples of the liquid culture can be automatically taken from the individual microreactors during the reaction with the aid of the pipetting robot at different points in time, which are analyzed offline with regard to certain process parameters. It is also possible to add fabric.
  • chemometric models can be created with little expenditure of time from the 2D fluorescence spectra recorded at the different points in time of the sampling.
  • mathematical and / or statistical relationships between the offline analyzed process parameters as well as the 2D fluorescence spectra of the liquid cultures recorded at the different times of sampling in the individual microreactors The chemometric model then enables process parameters of the liquid culture to be determined from a 2D fluorescence spectrum.
  • the data density can be increased by recording and evaluating the 2D fluorescence spectra of the liquid cultures recorded in these microreactors using a time vector.
  • reaction in the liquid cultures take place in several microreactors under matching conditions, the initial values of the process parameters to be recorded, for example the concentrations of Nutrients or (by-) products in which several microreactors are different.
  • the influence of the different initial values on the recorded 2D fluorescence spectra is used to develop the chemometric model.
  • Another possibility for creating a chemometric model without offline analysis of samples of the liquid cultures is that reactions in the cultures take place in several microreactors under identical conditions, with an analyte in the liquid cultures in each of the aforementioned microreactors at different times is added, which changes the process parameter to be recorded in the culture in a defined manner, so that, for example, known concentration jumps result. This means that the reactions in the different microreactors no longer take place under the same conditions. The influence of the defined change in the process parameter on the recorded 2D fluorescence spectrum of the respective liquid culture is then used to create the chemometric model.
  • process parameters for the calibration consists in describing the functional relationship on which the change in a process parameter in one of the liquid cultures is based by a mechanistic-mathematical model.
  • model parameters are assumed for the mechanistic-mathematical model, which may be wrong.
  • the process parameters determined on the basis of the mathematical model are compared with the 2D fluorescence spectra recorded at different times during the reaction and the model parameters are continuously corrected depending on the comparison. As a result, the process can be better mapped with each measured 2D fluorescence spectrum.
  • chemometric model there is also a mechanistic-mathematical model that describes the process well.
  • the concentration of individual substances can be determined on the basis of the recorded 2D fluorescence spectra determine with the help of chemometric models.
  • OTR oxygen transfer rate
  • the outlay on equipment usually required for the determination of respiratory activities is not necessary and the respiratory activity can be determined directly from the recorded 2D fluorescence spectra in the respective culture on the basis of a chemometric model previously determined by means of calibration.
  • illustration 1 shows a schematic structure of a device according to the invention for determining process parameters, such as substrate, protein and biomass concentration as well as substance transfer rates, by means of 2D fluorescence spectroscopy in a large number of different liquid cultures in microreactors of a shaken microtiter plate.
  • the device comprises a microreactor platform (4) connected to an orbital orbital shaker (5) on which at least one microtiter plate (2) with a plurality of microreactors (2a) is arranged. At least the bottom surfaces of the microreactors (2a) of the microtiter plate are transparent to electromagnetic radiation that is emitted and received by a measuring device.
  • the measuring device has a light source (8) and an automatically tunable monochromator (15).
  • the monochromator (15) is set up for the spectral isolation of different wavelengths from the incident light of the light source (8) in order to generate monochrome excitation light, the excitation wavelength of which is automatically changed step by step.
  • the measuring device further comprises a beam guidance system (3), which in the illustrated embodiment is formed by a Y-shaped optical waveguide (3a) and a coupling (3b).
  • the optical fibers of the two strands of the y-shaped optical waveguide (3a) converge in the area of the coupling (3b).
  • the beam guidance system (3) transmits the excitation light from the monochromator (15) to the liquid culture in one of the microreactors (2a) of the microtiter plate (2) and the emission spectrum from the liquid culture in the respective microreactor (2a) to an optical element (13) , for example a prism or grating.
  • the optical element (13) breaks down the emission spectrum (16) for each excitation wavelength into the different wavelengths and maps the fanned out (spread) emission spectrum on a sensor matrix (7), which is designed in particular as a CCD sensor.
  • the signals recorded by the sensor matrix are evaluated using a computer (6).
  • the coupling (3b) of the beam guidance system (3) is arranged at an acute angle of, for example, 35 degrees to the vertical on an xy positioning unit (1) and is aimed aligned with the transparent bottom surfaces of the individual microreactors (2a).
  • a shutter (14) which is used to interrupt the excitation light controlled by the computer (6) or the CCD camera comprising the sensor matrix (7) as a function of the position of the microreactor platform (4) is set up.
  • the position of the microreactor platform (4) is detected with a position transmitter (11), which in the illustrated embodiment is formed by a magnet (11b) arranged on the shaft of the orbital shaker and a Hall sensor (11a).
  • a position transmitter which in the illustrated embodiment is formed by a magnet (11b) arranged on the shaft of the orbital shaker and a Hall sensor (11a).
  • a Hall sensor 11a
  • Figure 2 illustrates how the emission spectrum (16) from the optical waveguide (3a) is coupled into the optical element (13).
  • the optical element (13) separates the emission spectrum (16) into its individual wavelengths and spreads it over the sensor matrix (7).
  • This optical arrangement creates bands on the sensor matrix (7) for the individual wavelengths and the intensity of the emission spectrum at the different wavelengths can be determined with the aid of the sensor matrix (7).
  • the illustrated structure of the optical element (13) and the sensor matrix (7) enables the simultaneous recording of a complete emission spectrum after the liquid culture has been excited by an excitation radiation with an excitation wavelength. By simultaneously capturing the intensities of the different wavelengths each Emission spectrum results in a considerable time saving in the measurement.
  • the recording of the 2D fluorescence spectra is computer-aided with the aid of the shutter (14) and is synchronized with the position of the microreactor platform (4) and thus with the liquid culture in the respective microreactor (2a).
  • the optical path of the excitation light is released for a defined period with the aid of the mechanical shutter (14).
  • the synchronization ensures that the excitation light falls into the microreactor (2a), which rotates above the stationary coupling (3b), at a defined point in time and at a defined position.
  • the optical beam path of the excitation light is closed again by the mechanical shutter (14) so that no more excitation radiation can enter the microreactor (2a).
  • a significant advantage of the synchronization is that a defined measuring segment (18) of the microreactor (2a), as in Figure 3 shown schematically, can be examined and thus, for example, the liquid sickle (17) of the liquid culture rotating in the microreactor (2a) can be taken into account in a targeted manner.
  • the measuring segment (18) is the area into which the excitation light is coupled in a position-controlled manner into one of the microreactors (2a) of a shaken microtiter plate (2) when measuring an emission spectrum.
  • FIG 3 two measuring segments (18) are shown.
  • the measurement can be started specifically at any position during the rotation of the microreactor (2a) above the coupling (3b).
  • the integration time of the measurement signal segments of the sensor matrix (7) By varying the integration time of the measurement signal segments of the sensor matrix (7), measurement signals of different lengths can be examined within each microreactor (2a).
  • the measuring segment (18) extends out of the empty microreactor (2a) into the liquid sickle (17), whereby the emission spectrum can be falsified.
  • the measuring segment (18) lies exclusively within the liquid sickle (17), so that only the emission spectrum of the liquid culture is recorded during the measurement, which is to be aimed for.
  • the position of the coupling (3b) relative to the shaken microtiter plate (2) is selected such that the center of the rotational movement of the microtiter plate (2) lies exactly between several microreactors (2a), as shown in FIG Figure 4B for two microreactors and in Figure 4C for four microreactors is shown. Since the electromagnetic radiation (19) of the excitation light per revolution of the orbital shaker (5) with a corresponding Figure 4 is introduced into several microreactors (2a) one after the other, several microreactors (2a) can be measured without repositioning the coupling (3b).
  • the xy positioning unit (1) In order to measure all of the microreactors (2a) of a microtiter plate (2), fewer movements of the coupling (3b) with the aid of the xy positioning unit (1) are required, which means that the measurement can be significantly accelerated. If the coupling (3b), for example, as in Figure 4C shown, positioned in the center of four neighboring microreactors (2a), the xy positioning unit (1) only has to move to 24 instead of 96 positions to measure all microreactors of a 96-well microtiter plate. This saves a considerable amount of time when measuring all of the microreactors (2a) of the microtiter plate (2).
  • the assignment of the emission spectra from the respective microreactors (2a) during one rotation of the microreactor platform (4) can be carried out using the position transmitter (11).
  • the position transmitter (11) detects which microreactor (2a) is in the focus of the coupling (3b) at which point in time of the rotation.
  • Another advantage of several microreactors (2a) rotating over an optical coupling (3b) is that larger shaking diameters (preferably 6mm, 9mm, 12.5mm or larger) can be used. Larger shaking diameters allow lower speeds of the orbital shaker (5) in order to generate a rotating fluid sickle (17) in the microreactors (2a).
  • larger shaking diameters are particularly suitable for examining Liquid cultures with increased viscosity, such as those that can arise when cultivating biopolymers or filamentous organisms, for example.
  • the intensity of the excitation light and thus the intensity of the emission spectrum can be increased and ultimately the sensitivity of the measuring device can be improved.
  • the movement of the optical element (13) takes place as a function of the excitation wavelength of the introduced excitation light.
  • the assignment of the wavelength to the position (pixel) on the sensor matrix (7) must be recalculated with this method using the wavelength of the excitation light.
  • Figure 5B shows an alternative embodiment for reducing the backscatter.
  • a screen (20) is arranged between the optical element (13) and the sensor matrix (7), which fades out the light of the respective excitation wavelength so that it does not hit the sensor matrix (7).
  • the diaphragm (20) is preferably moved by means of a stepping motor when each excitation wavelength is introduced in such a way that the optical path of the exciting wavelength between the optical element (13) and the sensor matrix (7) is blocked.
  • This structure enables a higher intensity of the excitation light and thus a higher intensity of the emission spectrum, which leads to stronger fluorescence signals and ultimately improved measured values.
  • Figure 6B shows the use of additional optically active components, such as a collimator (21), which parallelizes the electromagnetic radiation (19) of the excitation light.
  • the collimator (21) is attached to the end of the optical waveguide (3a) in the area of the coupling (3b). The parallelization allows the excitation light to be introduced into the liquid cultures with a higher energy intensity.
  • the collimator (21) can also bundle the coupled-in emission spectrum (16) in order to collect larger amounts of the emission spectrum.
  • Figure 5 shows a device in which the backscattering of the electromagnetic radiation (19) radiated into the liquid culture of a microreactor (2a) with a separate, in Figure 7 light-sensitive sensor, not shown, is detected.
  • the backscattered excitation light is fed to the light-sensitive sensor with a further optical waveguide (3c).
  • the additional optical waveguide (3c) is aligned in the coupling (3b) with an angle of incidence of, for example, 35 degrees to the vertical.
  • the angle of incidence of the optical waveguide (3a) for fluorescence measurement is in this case 0 degrees to the vertical.
  • This alignment of the optical waveguide (3a) in the coupling (3b) improves the focusing and recording of the emission spectrum.
  • the optical waveguides (3a, 3c) are aligned in the coupling (3b) in such a way that the focus of both optical waveguides lies in one point. Accordingly, the focus can be aligned exactly to the same position within the microreactor (2a).
  • Figure 8 shows an apparatus for carrying out the method accordingly illustration 1 , but with a differently structured beam guidance system (3).
  • the excitation light from the light source (8) is deflected by a semitransparent mirror (22) and introduced into the liquid culture in one of the microreactors (2a) via an optical waveguide (23) with only one fiber.
  • the emission spectrum (16) of the liquid culture is transmitted through the optical waveguide (23) and the semitransparent mirror (22) to the optical element (13) and finally to the sensor matrix (7).
  • the single fiber of the optical waveguide (23) is opposite to the fibers of the fiber bundle of the optical waveguide (3a) illustration 1 thicker. This has the result that the radius of the optical waveguide (23) as a whole (and thus the radius of the electromagnetic radiation) is reduced and the radiation can thus be better focused. The better focusing prevents crosstalk with neighboring microreactors.
  • microtiter plates (2) can be arranged together on a microreactor platform (4), the microreactors (2a) of all microtiter plates (2) being measured sequentially.
  • the coupling (3b) is no longer merely moved from microreactor (2a) to microreactor (2a) below a microtiter plate (2), but rather below several microtiter plates (2). If, for example, four 48-well microtiter plates are arranged on the microreactor platform (4), a total of 192 parallel cultivations can be carried out and measured.
  • FIG. 9 shows Figure 9 a device with which 2D fluorescence spectra of the liquid cultures in different microreactors (2a) of several microtiter plates (2) with several measuring devices can be recorded at the same time.
  • the optical waveguides (3a) for fluorescence measurement and the optical waveguides (3c) for measuring the backscatter are arranged on movable arms of an xy positioning unit (1) in such a way that microreactors (2a) can be measured on different microtiter plates (2) at the same time.
  • the microreactors (2a) of different microtiter plates (2) are approached synchronously and simultaneously. That is, after each positioning, three microreactors (2a) are measured simultaneously in the example shown, the microreactors being located in the different microtiter plates (2) at corresponding positions. Since all microtiter plates (2) are on the microreactor platform (4) of an orbital shaker (5), the measurements can be started at the same time and synchronized by means of a single position encoder (11).
  • Figure 10 shows a further possibility for increasing the rate of acquisition of the fluorescence spectra in a Microtiter plate (2a).
  • the optical couplings (3b) of several measuring devices are attached to the arm of a positioning unit (1) at an identical distance from the centers of the microreactors (2a) and can be moved between the microreactors (2a) of a single microtiter plate (2).
  • a positioning unit (1) Assuming a corresponding number of coupling devices (3b) and measuring devices, complete rows or columns of microreactors (2a) of a microtiter plate (2) can be measured simultaneously.
  • the positioning unit only has to move the coupling (3b) in one direction in this case in order to completely measure a microtiter plate.
  • the fluorescence and / or backscattering can be measured simultaneously in a complete row of the microtiter plate (2).
  • the optical waveguides (3a, 3c) therefore only have to be moved in one dimension by means of the positioning unit (1). The associated time savings lead to an increased measurement frequency and data density.
  • the device comprises a pipetting robot to automatically take samples of the liquid cultures from the microreactors (2a) at different times during the cultivation or to add water or solutions with, for example, nutrients or (by-products).
  • a pipetting robot to automatically take samples of the liquid cultures from the microreactors (2a) at different times during the cultivation or to add water or solutions with, for example, nutrients or (by-products).
  • the combination of the method according to the invention with the automatic Sampling enables accelerated creation of chemometric models.
  • Samples for offline analysis can be automatically taken from the liquid culture from the ongoing cultivation at different times.
  • Chemometric models can be created from the offline analyzed process parameters of the samples and the 2D fluorescence spectra recorded at the different times when the sample was taken or added.
  • FIG 11 a process management is shown schematically in which in several microreactors (well 1 to well 3) cultivations of liquid cultures take place under identical conditions. From each of the aforementioned liquid cultures in wells 1-3, 2D fluorescence spectra (measurement) are recorded at different times.
  • the microreactor (well 1) for example, the measurements 1.1 to 1.3
  • the microreactor (well 2) the measurements 2.1 to 2.3
  • the microreactor (well 3) the measurements 3.1 to 3.3.
  • the 2D fluorescence spectra 1.1 to 3.3 recorded with a time offset in wells 1 to 3 are combined in such a way that the 2D fluorescence spectra from the different wells 1 to 3 are recorded over a common time vector.
  • the wave-related resolution of the measurements is lost, but the time intervals between two measurements can be significantly reduced.
  • Figure 12 shows a procedure accordingly Figure 11 , in which a further increase in the data density can be achieved in that the microreactor (here: well 1) is no longer measured after the reaction liquid has been removed. Instead of an unnecessary measurement in the microreactor (well 1) after the reaction liquid has been removed, a measurement is taken directly in the microreactor (well 2) in this procedure. This means that another measurement (measurement 2.4) can be carried out in well 2 within 5.63 minutes. Accordingly, nine usable measurements are available for creating the chemometric model.
  • Figure 13B illustrates the conventional procedure for creating chemometric models based on 2D fluorescence spectra in individual stirred tank fermenters ( Figure 13A ) a method in which in several microreactors (2a) a microtiter plate (2) cultivations in the liquid cultures under matching Conditions run, the initial values of the process parameter to be recorded (eg concentration of the carbon source, products or by-products) in the liquid cultures of the individual microreactors are different.
  • the process parameter to be recorded eg concentration of the carbon source, products or by-products
  • this process management is associated with an unacceptable expenditure of time and experimentation.
  • due to the parallel cultivation in the microreactors of the microtiter plate according to the present invention such an approach is possible without problems.
  • Figure 14 illustrates an alternative method for creating chemometric models based on 2D fluorescence spectra.
  • Several cultivations with matching starting conditions are carried out in parallel in microreactors on a shaken microtiter plate and monitored by recording 2D fluorescence spectra.
  • an analyte is added to selected microreactors so that known concentration jumps result. This means that the reactions in the selected microreactors no longer take place under the same conditions.
  • These changes are made possible by the continuous recording of 2D fluorescence spectra in the microreactors Microtiter plate detected.
  • the influence of the defined changes on the recorded 2D fluorescence spectra is used to develop chemometric models. But the dilution effects resulting from the addition of water can also be used to develop chemometric models.
  • the addition of water to the culture broth also causes known concentration drops, which are detected by means of the 2D fluorescence spectra.
  • the OTR can also be determined on the basis of the recorded 2D fluorescence spectra with the aid of chemometric models.
  • the method according to the invention and the device for carrying out the method thus make it possible for the first time to measure the oxygen transfer rate resolved by individual microreactors of a microtiter plate on the basis of 2D fluorescence spectra.
  • the pH can be determined using 2D fluorescence spectra in combination with chemometric models.
  • the state of the art for determining pH is the use of optodes or dyes in microtiter plates. Optodes or dyes are not required for pH determination with the present invention.
  • E. coli is one of the fastest growing microorganisms used in biotechnology. Even with this high growth rate, the presented system can generate a sufficiently high data density to be able to follow the courses of different process-relevant concentrations as well as the pH value.

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Claims (38)

  1. Procédé, destiné à déterminer des paramètres de processus au moyen de la spectroscopie de fluorescence en 2D dans des milieux de cultures liquides dans plusieurs microréacteurs (2a) d'au moins une plaque de microtitration, que l'on agite en continu dans tous les microréacteurs à l'aide d'un agitateur orbital (5) au moins jusqu'à l'achèvement de la culture, pendant la culture, les spectres de fluorescence en 2D des milieux de cultures liquides étant enregistrés par au moins un dispositif de mesure désaccouplé du mouvement d'agitation de la plaque de microtitration, comportant les étapes suivantes, consistant à :
    1.1 générer une lumière d'excitation monochrome, dont la longueur d'onde d'excitation est modifiée graduellement,
    1.2 introduire successivement de la lumière d'excitation avec différentes longueurs d'onde d'excitation dans le milieu de culture liquide dans l'un des microréacteurs,
    1.3 transmettre les spectres d'émission du milieu de culture liquide dans le microréacteur vers un élément optique, lequel décompose le spectre d'émission pour chaque longueur d'onde d'excitation dans les différentes longueurs d'onde et le reproduit en éventail sur une matrice de capteurs (7) pourvue de capteurs photosensibles,
    1.4 enregistrer le spectre de fluorescence en 2D par détection de l'intensité des différentes longueurs d'onde pour chaque spectre d'émission au moyen de la matrice de capteurs, et
    1.5 enregistrer les spectres de fluorescence en 2D des milieux de cultures liquides dans d'autres microréacteurs de l'au moins une plaque de microtitration, par les étapes 1.1 à 1.4.
  2. Procédé selon la revendication 1, caractérisé en ce que l'introduction de la lumière d'excitation et la transmission des spectres d'émission s'effectuent à travers une surface transparente à la lumière d'excitation et aux spectres d'émission sur la face inférieure de chaque microréacteur.
  3. Procédé selon la revendication 1 ou 2, caractérisé en ce que la lumière d'excitation est générée à l'aide d'un monochromateur (15) automatiquement accordable, pour l'isolation spectrale de différentes longueurs d'onde à partir de la lumière incidente d'une source lumineuse (8).
  4. Procédé selon la revendication 3, caractérisé en ce que la transmission de la lumière d'excitation du monochromateur vers le milieu de culture liquide et la transmission du spectre d'émission du milieu de culture liquide vers l'élément optique s'effectuent à l'aide d'un système de guidage de faisceau (3) comprenant un coupleur optique (3b), pour l'introduction de la lumière d'excitation dans le milieu de culture liquide, ainsi que pour l'injection du spectre d'émission dans le système de guidage de faisceau, le coupleur optique étant orienté sur le microréacteur contenant le milieu de culture liquide.
  5. Procédé selon la revendication 4, caractérisé en ce que pendant l'enregistrement du spectre de fluorescence en 2D, l'on ne fait pas bouger le coupleur optique, de sorte que les microréacteurs agités bougent par rapport au coupleur optique.
  6. Procédé selon la revendication 4 ou 5, caractérisé en ce que suite à l'enregistrement du spectre de fluorescence en 2D, le coupleur optique est déplacé par une unité de positionnement (1) entre les microréacteurs de la (des) plaque(s) de microtitration.
  7. Procédé selon l'une quelconque des revendications 3 à 6, caractérisé en ce que le diamètre d'agitation de l'agitateur orbital est adapté de telle sorte que pendant l'enregistrement du spectre de fluorescence, la lumière d'excitation soit introduite exclusivement dans le milieu de culture liquide de l'un des microréacteurs et que le spectre d'émission dudit milieu de culture liquide soit introduit exclusivement dans le coupleur optique.
  8. Procédé selon l'une quelconque des revendications 1 à 7, caractérisé en ce que les spectres de fluorescence en 2D des milieux de cultures liquides sont enregistrés simultanément dans différents microréacteurs à l'aide de plusieurs dispositifs de mesure.
  9. Procédé selon la revendication 8, caractérisé en ce que les coupleurs optiques des plusieurs dispositifs de mesure sont déplaçables à l'aide d'une unité de positionnement commune entre les microréacteurs de la (des) plaque(s) de microtitration.
  10. Procédé selon l'une quelconque des revendications 4 à 6, caractérisé en ce que le diamètre d'agitation de l'agitateur orbital est adapté de telle sorte que pendant un tour de l'agitateur orbital, plusieurs microréacteurs tournent successivement au-dessus du coupleur optique d'un dispositif de mesure, les spectres de fluorescence enregistrés étant associés aux microréacteurs qui tournent au-dessus du coupleur optique.
  11. Procédé selon l'une quelconque des revendications 1 à 10, caractérisé en ce que l'introduction de la lumière d'excitation est interrompue en fonction de la position de l'agitateur orbital.
  12. Procédé selon la revendication 11, caractérisé en ce que la position de l'agitateur orbital est déterminée à l'aide d'un détecteur de position.
  13. Procédé selon l'une quelconque des revendications 1 à 12, caractérisé en ce que la longueur d'onde d'excitation dans le spectre d'émission est masquée.
  14. Procédé selon la revendication 13, caractérisé en ce que par une modification ciblée de la position de l'élément optique, on fait passer à l'avant de la matrice de capteurs le domaine du spectre d'émission dont la longueur d'onde est inférieure ou égale à la longueur d'onde d'excitation.
  15. Procédé selon la revendication 13, caractérisé en ce que la longueur d'onde d'excitation est masquée par un diaphragme déplaçable entre l'élément optique et la matrice de capteurs.
  16. Procédé selon l'une quelconque des revendications 1 à 15, caractérisé en ce qu'avant l'introduction dans le milieu de culture liquide, la lumière d'excitation est parallélisée ou focalisée et/ou le spectre d'émission est concentré.
  17. Procédé selon l'une quelconque des revendications 1 à 16, caractérisé en ce que la rétrodiffusion de la lumière d'excitation irradiée dans le milieu de culture liquide est détectée par un capteur photosensible séparé du dispositif de mesure.
  18. Procédé selon l'une quelconque des revendications 4 à 17, caractérisé en ce que la lumière d'excitation et le spectre d'émission sont transmis dans le système de guidage de faisceau par l'intermédiaire de guides d'ondes optiques séparés ou d'un guide d'ondes optiques en Y disposant de fibres séparées pour la lumière d'excitation et le spectre d'émission.
  19. Procédé selon l'une quelconque des revendications 4 à 17, caractérisé en ce que dans le système de guidage de faisceau
    - la lumière d'excitation est déviée par un miroir semi-transparent et introduite par l'intermédiaire d'un guide d'ondes optiques doté d'une seule fibre dans le milieu de culture liquide, et
    - le spectre d'émission est transmis via le guide d'ondes optiques et le miroir semi-transparent vers l'élément optique.
  20. Procédé selon l'une quelconque des revendications 1 à 19, caractérisé en ce que pendant la culture, à l'aide d'un robot de pipetage, en différents moments, des échantillons du milieu de culture liquide lesquels sont analysés hors ligne au niveau de paramètres de processus déterminés sont pris dans l'un des microréacteurs, et/ou à l'aide du robot de pipetage, en différents moments, des substances et/ou des liquides sont automatiquement ajoutés au milieu de culture liquide.
  21. Procédé selon la revendication 20, caractérisé en ce que les paramètres de processus des échantillons analysés hors ligne, ainsi que les spectres de fluorescence en 2D enregistrés en différents moments sont utilisés pour établir des modèles chimiométriques.
  22. Procédé selon l'une quelconque des revendications 1 à 21, caractérisé en ce que
    - dans plusieurs microréacteurs, des cultures de milieux de cultures liquides se déroulent dans des conditions concordantes,
    - dans chacun des milieux de cultures liquides précédemment cités, des spectres de fluorescence en 2D sont enregistrés avec un décalage temporel,
    - les spectres de fluorescence en 2D enregistrés respectivement avec un décalage temporel dans les plusieurs microréacteurs sont regroupés de telle sorte que les spectres de fluorescence provenant des microréacteurs précédemment cités soient détectés via un vecteur de temps.
  23. Procédé selon l'une quelconque des revendications 1 à 19, caractérisé en ce que dans plusieurs microréacteurs, des cultures dans les milieux de cultures liquides se déroulent sous des conditions concordantes, les valeurs de départ du paramètre de processus qui doit être détecté dans les milieux de cultures liquides étant différentes dans les microréacteurs et l'influence des différentes valeurs de départ sur les spectres de fluorescence en 2D enregistrés étant utilisée pour développer des modèles chimiométriques.
  24. Procédé selon l'une quelconque des revendications 1 à 19, caractérisé en ce que dans plusieurs microréacteurs, des cultures dans les milieux de cultures liquides se déroulent sous des conditions concordantes, en différents moments étant ajoutés aux milieux de cultures liquides de microréacteurs individuels parmi ceux précédemment cités une substance et/ou un liquide, qui modifie de manière définie le paramètre de processus qui doit être détecté dans les milieux de cultures liquides, et l'influence de la modification définie sur les spectres de fluorescence en 2D enregistrés est utilisée pour développer des modèles chimiométriques.
  25. Procédé selon l'une quelconque des revendications 1 à 19, caractérisé en ce que
    - la relation fonctionnelle sur laquelle est basée la modification d'un paramètre de processus dans l'un des milieux de cultures liquides est décrite par un modèle mathématico-mécaniste,
    - au début de la culture, des paramètres de modèle sont supposés pour le modèle mathématique,
    - les paramètres de processus déterminés à base du modèle mathématique sont comparés avec les spectres de fluorescence en 2D enregistrés en différents moments pendant la culture dudit milieu de culture liquide et
    - les paramètres de modèle sont corrigés en fonction de la comparaison.
  26. Procédé selon l'une quelconque des revendications 21 à 25, caractérisé en ce qu'à l'aide des modèles chimiométriques, au moins un paramètre de processus est déterminé à l'aide d'un spectre de fluorescence en 2D enregistré par un milieu de culture liquide.
  27. Dispositif, destiné à détecter des paramètres de processus par spectroscopie de fluorescence en 2D dans des milieux de cultures liquides dans plusieurs microréacteurs d'une plaque de microtitration agitée, comprenant :
    - une plate-forme de microréacteurs (4) reliée avec l'agitateur orbital (5) sur laquelle est placée au moins une plaque de microtitration pourvue de plusieurs microréacteurs,
    - une source lumineuse (8),
    - un monochromateur (15) automatiquement accordable pour l'isolation spectrale de différentes longueurs d'onde à partir de la lumière incidente de la source lumineuse, le monochromateur étant aménagé pour générer de la lumière d'excitation monochrome et pour modifier graduellement sa longueur d'onde d'excitation,
    - un élément optique (13) et un système de guidage de faisceau (3) comprenant un coupleur optique (3b), qui est aménagé pour transmettre la lumière d'excitation du monochromateur vers le milieu de culture liquide et pour transmettre le spectre d'émission du milieu de culture liquide vers l'élément optique pendant un mouvement d'agitation continu de la plateforme de microréacteurs, assuré par l'agitateur orbital,
    - pour l'introduction de la lumière d'excitation dans le milieu de culture liquide, ainsi que pour l'injection du spectre d'émission dans le système de guidage de faisceau, le coupleur optique étant orienté sur un segment du microréacteur qui est transparent au rayonnement électromagnétique et
    - l'élément optique décomposant le spectre d'émission pour chaque longueur d'onde d'excitation dans les différentes longueurs d'onde et le déployant en éventail,
    - une matrice de capteurs (7), dotée de capteurs photosensibles, qui est placée de telle sorte que l'élément optique reproduise le spectre d'émission déployé en éventail sur la matrice de capteurs,
    - la matrice de capteurs étant aménagée pour enregistrer un spectre de fluorescence en 2D, par détection de l'intensité des différentes longueurs d'onde pour chaque spectre d'émission.
  28. Dispositif selon la revendication 27, caractérisé en ce que le coupleur optique est déplaçable par une unité de positionnement (1) entre les microréacteurs de la (des) plaque(s) de microtitration.
  29. Dispositif selon la revendication 27 ou 28, caractérisé en ce que
    - le dispositif comporte plusieurs dispositifs de mesure qui comportent une source lumineuse, un monochromateur automatiquement accordable, un système de guidage de faisceau, un élément optique, ainsi qu'une matrice de capteurs,
    - et les spectres de fluorescence en 2D des milieux de cultures liquides sont détectés simultanément dans plusieurs microréacteurs par les plusieurs dispositifs de mesure.
  30. Dispositif selon la revendication 29, caractérisé en ce que les coupleurs optiques des plusieurs dispositifs de mesure sont déplaçables par une unité de positionnement commune entre les microréacteurs de la(des) plaque(s) de microtitration(n).
  31. Dispositif selon l'une quelconque des revendications 27 à 30, caractérisé en ce que le dispositif comporte un obturateur placé dans le trajet optique de la lumière d'excitation, qui est aménagé pour interrompre la lumière d'excitation en fonction de la position de l'agitateur orbital.
  32. Dispositif selon la revendication 31, caractérisé en ce que sur l'agitateur orbital est placé un détecteur de position destiné à détecter la position de l'agitateur orbital et en ce qu'un système de commande est aménagé pour traiter le signal de position détecté par le détecteur de position, ainsi que pour interrompre la lumière d'excitation en fonction des signaux de position, au moyen de l'obturateur.
  33. Dispositif selon l'une quelconque des revendications 27 à 32, caractérisé en ce qu'entre l'élément optique et la matrice de capteurs est placé un diaphragme déplaçable, destiné à masquer la longueur d'onde d'excitation.
  34. Dispositif selon l'une quelconque des revendications 27 à 33, caractérisé en ce sur le coupleur est placée une lentille pour la collimation ou pour la focalisation de la lumière d'excitation.
  35. Dispositif selon l'une quelconque des revendications 27 à 34, caractérisé en ce que le dispositif comporte un capteur photosensible, aménagé pour détecter la rétrodiffusion de la lumière d'excitation irradiée dans le milieu de culture liquide.
  36. Dispositif selon l'une quelconque des revendications 27 à 35, caractérisé en ce que le système de guidage de faisceau comporte des guides d'ondes optique séparés ou un guide d'ondes en Y, disposant de fibres séparées.
  37. Dispositif selon l'une quelconque des revendications 27 à 35, caractérisé en ce que le système de guidage de faisceau comporte en tant que composants optiques un miroir semi-transparent et un guide d'ondes optiques doté d'une seule fibre, les composants optiques étant placés les uns par rapport aux autres de telle sorte que la lumière d'excitation soit déviée par le miroir semi-transparent et soit introduite via le guide d'ondes optique dans le milieu de culture liquide microbien et en ce que le spectre d'émission est transmis à travers le guide d'ondes optiques et le miroir semi-transparent vers l'élément optique.
  38. Dispositif selon l'une quelconque des revendications 27 à 37, caractérisé en ce que le dispositif comprend un robot de pipetage, aménagé pour prélever automatiquement dans un microréacteur, en différents moments pendant la culture des échantillons du milieu de culture liquide microbien ou pour ajouter des substances et/ou des liquides.
EP17757504.0A 2016-09-01 2017-08-16 Procede et dispositif permettant d'enregistrer des parametres de processus de cultures liquides Active EP3458844B1 (fr)

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ES2871779T3 (es) 2021-11-02
EP3458844A1 (fr) 2019-03-27
DK3458844T3 (en) 2021-05-10
CN109642870B (zh) 2023-05-12
DE102016116377A1 (de) 2018-03-01
JP7057349B2 (ja) 2022-04-19
US11635381B2 (en) 2023-04-25
CN109642870A (zh) 2019-04-16
JP2019532278A (ja) 2019-11-07
WO2018041634A1 (fr) 2018-03-08
PL3458844T3 (pl) 2021-10-18

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